H. capsulatum is a dimorphic pathogenic fungus responsible for histoplasmosis, the most common systemic mycosis in the region of the Ohio-Mississippi river valley in the Midwestern United States. Severe disease is rare in endemic regions in the immunocompetent individuals, but a life- threatening disseminated mycosis occurs in those patients with AIDS or in individuals receiving immunosuppressive therapy. H. capsulatum exists in two morphologically distinct phases which are controlled by environmental temperatures and other factors: the yeast phase is, however, found exclusively in infected tissues and is the form required for progressive infection. Clinical isolates of H. capsulatum vary with respect to virulence in murine models and in their in vitro temperature sensitivity. Four distinct H. cpasulatum genes have been cloned by a subtraction library strategy that are expressed in the yeast phase and which are transcriptionally silent in the mycelial forma of virulent strains. This group of yeast phase specific (yps) genes are not expressed in the low virulence, temperature sensitive Dawns strains, although the genes and their putative regulatory regions are retained in the Downs genome. These findings are most consistent with the regulation of H. capsulatum gene expression during the mycelial to yeast transition or in the yeast phase by synthesis of temperature inducible transcription factors. Using the regulatory regions of the yps genes as targets, this project explores temperature sensitive transcriptional regulation in H. capsulatum via the analysis of master regulatory proteins, including p30, a novel 30 kD protein found in high levels in nuclear extracts from mycelial forms of virulence Hc strains which binds to the 5' regulatory regions of at least two yps genes. The p30 protein binds at greatly reduces levels in Southwestern blots from extracts from the yeast phase of highly virulent organisms, but the Downs strain is deficient in the expression or synthesis of the regulatory protein. The project will examine the specific DNA regions protected in the 5' target sequences by the p30M nuclear protein and will seek to confirm a putative decanucleotide (TCCTTTTTTT) motif implicated as a binding site. Using DNA affinity chromatography, the p30M nuclear binding protein will be isolated and characterized and limited amino acid sequence will be obtained to generate degenerate oligonucleotide. These reagents will be used to isolate the p30 gene from genomic or cDNA libraries which will then be characterized at the molecular level. The biologic activity of the cloned p30M gene will be explored by introducing it into various H. capsulatum strains differing thermotolerance and virulence. The studies should provide significant insight into the mechanism by which master regulatory proteins govern gene transcription in a pathogenic fungus where a temperature sensitive switch influences morphology and virulence.